The goal of this project is to develop methods for manufacturing den- dritic cells for clinical immunotherapy trials. Over the past year, we have developed and optimized a full-scale GMP method for 5-day flask culture of autologous dendritic cells in RPMI, autologous plasma or allogeneic serum, IL4, and GMCSF, starting with peripheral blood monocytes collected by apheresis and purified by elutriation. The dendritic cells generated are then available for further manipulations (e.g., peptide pulsing) prior to clinical administration. To date, this manufacturing method has been implemented in two clinical trials, one for pediatric sarcoma and one for colon cancer. Further studies are aimed at improving the closed system aspects of the method by substituting bags for flasks as culture containers: an evaluation of three novel bags from two different manufacturers is in pro-gress. This project has required simultaneous development and evaluation of assays for quantitation and characterization of these cell populations. Extensive flow cytometric phenotyping of these cultured cells has led to the development of a preliminary basic flow cytometric panel that will be evaluated in the context of clinical trials. This panel assesses forward scatter, side scatter, 7AAD viability, CD14, mannose receptor, CD1a, CD80, and CD83 of the cultured dendritic cells.